Thrombin induces the rapid formation of inositol bisphosphate and inositol trisphosphate in human platelets

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Thrombin induces the rapid formation of inositol bisphosphate and inositol trisphosphate in human platelets Wolfgang

Siess and Hedge Bmder

MedzzznzscheKlznzk Innenstadt der Unzversrtat, Zzemssenstr I,8000 Munchen 2, FRG

Recetved 9 November 1984 Human platelets prelabeled wtth [3H]mosttol were exposed to thrombm The aqueous soluble mosttol phosphates were separated by amon exchange column chromatography, paper chromatography or high-performance hqutd chromatography, and ldentl~ed by c~hromatography wtth authentic standard substances Thrombm tmmedtately mduces the raped formatton of mosrtol 1,4-btsphosphate and mosttol 1,4,5-trtsphosphate Accumulation of mosttol-1-monophosphate and mositol-2-monophosphate occurs later after a time lag of 10 set The results mdtcate that the phosphohpase C Induced polyphosphomosrttde hydrolyses rather than the phosphatldyllnosltol hydrolyses is the tnggermg event for platelet actrvatron, and support the concept of mosttol I ,4,Stnsphosphate as putattve second messenger Piatelet actzvatzon

Thrombzn

Phospholzpase C

1. INTRODUCTION Physlologic~ platelet strmuh such as thrombin, collagen, ADP, platelet-activating factor, arachrdonic acid and prostaglandin endoperoxides induce m platelets the degradation of inositol containing phospholipids [l-6]. This reaction, which is catalysed by phospholipase C, leads to two putative second messengers: mositol 1,4,5-trisphosphate, which can mobihse Ca2+ from nonmltochondrlal pools, and 1,2-diacylgly~erol, which stimulates protein phosphorylation by protem kinase C activation [7-91. Both molecules have been found in stimulated platelets, and they may act synergistically to induce the physlologic~ platelet response [2,10,11]. Recently, we have shown the close associatton of phospholipase C activation with each physiological platelet response, i.e., platelet shape change, release reaction and aggregation [5,6,12-151. It is not clear which of the Abbrevzatzons: PtdIns,

phosphatldyllnosltol; PtdIns4P, phosphat~dyllnositol 4-phosphate; PtdIns4,5Pz, phosphattdylinositol 4,S-bisphosphate; HPLC, high-perfor-

mance liquid chromatography

Znosztolphosphate

Znosztolphospholzpzd

three inositol phospholipids - PtdIns, PtdIns4P or PtdIns4,5Pz - is degraded initially by phospholipase C upon receptor activation [ 161. Following recent studies on platelets and other cell types it seems that PtdIns4JPz rather than PI is hydrolysed ([10,17-191; for review see [7]). To address this question further, we measured the formation of the respective inositol phosphates m human platelets upon activation with thrombin. 2. MATERIALS

AND METHODS

2.1. Materials L-myo[2-3H]inositol was purchased from NEN, and L-3-phosphatidyl[U-14C]inositol and Lmyo[U-‘4CJinositol-l-phosphate were from Amersham. L-cY-phosphatidylinositol, L-cu-phosphatidylinositol 4-monophosphate, Lti-phosphatidylinositol4,5-bisphosphate, phosphoinositides, L-cuglycerophosphate, Dowex 1 x 8 (chloride form 100-200 mesh), Dowex-HCR-W2, myoinositol 2-monophosphate and phospholipase C from Bacillus cereus were all obtained from Sigma. Equipment and columns for HPLC were from Waters.

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2.2. Preparation of mositol phosphate standards The alkaline hydrolysis of phosphomositides as described by Grado and Ballou [20] leads to a mixture of two isomers of inosrtol trisphosphate and of inositol bisphosphate which are separated by descending paper chromatography [21]. By comparison with the work of Ballou and colleagues [20,22] and Irvine et al. [23], these compounds are identified as inositol 1,4,5trisphosphate (slowrunning), mositol 2,4,5trisphosphate (fastrunning), inositol 1,Cbrsphosphate (slow-running) and inosrtol 4,5-bisphosphate (fast-running). Inosrtol 1,4,5trrsphosphate and mositol 1,Cbisphosphate were also obtained by alkaline hydrolysis of authentic PtdIns4,5P2 and PtdIns4P. Glycerophosphoinositol was prepared by mild alkaline hydrolysis from phosphatidylinosrtol [24]. Inositol 1,2-cyclic monophosphate was obtained by incubation of phosphatidylinositol with phospholipase C from platelet cytosol or from B. cereus [25,26]. Inositol 1-monophosphate and inositol-2-monophosphate were obtained by heating inositol 1,2cychc monophosphate at 100°C for 15 min in 0.1 N HCl [27]. 2.3. Preparation of platelets labeled with PHjinositol Platelet rich plasma was prepared from human blood (100 ml) anticoagulated with acidic citrate/dextrose [ 121. Prostacyclin (300 ng/ml) was added, platelets were centrifuged and resuspended in Tyrode-Hepes buffer [12] at a platelet concentration of 2-2.5 x 109/ml. [3H]Inositol(1 mCi) and MnCl2 (3 mM) were added, and platelet suspensions were incubated m aggregometer tubes at 37°C for 90 min while stirring (250 rpm). PGEr (1 fig/ml) and EGTA (5 mM) were added, and after further 30 min of incubation the platelets were centrifuged and washed once m Tyrode-Hepes buffer containing 5 mM unlabeled inositol, 1 mM EGTA, 10% of plasma and prostacyclin (300 ng/ml). Platelets were finally resuspended in lo-12 ml of Tyrode-Hepes buffer containing 1 mM EGTA. Samples of platelet suspensions (0.5 ml) were placed in aggregometer tubes, LiCl (10 mM) was added, and platelets were stirred for 2 mm before exposure to thrombin (2 U/ml). Shape change and aggregation were recorded. Reactions were stopped by transferring the samples into 1.8 ml of 108

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chloroform/methanol, 1: 2. Since endogenously formed mositol phosphates including inositol trtsphosphate were recovered at the same rate following extraction at acidic or neutral pH, aqueous soluble inositol phosphates were extracted at neutral pH [28] in order to avoid degradation of mositol 1,2-cyclic monophosphate [27]. The samples were then frozen, lyophilised and stored at - 20°C. 2.4. Separation of rnosltol phosphates Amon exchange column chromatography on Dowex 1 x 8 columns (1 ml) was used as first step to separate the inosttol phosphates. Dowex in chloride form was converted mto the formate form with 8 ml of 5% formic acid and rinsed with 25 ml of H20. The lyophihsed platelet samples were dissolved in 1 ml of Hz0 and a mixture of unlabeled inosttol phosphate standards (inosnol 1,4,5_trisphosphate, inositol 1,4-brsphosphate, glycerophosphoinosttol, mositol l-phosphate, mositol2-phosphate) was added. If the samples were separated subsequently on HPLC, two fractions were eluted: Inositol and some glycerophosphomositol in 30 ml of Hz0 and mosrtol phosphates m 14 ml of 1 M ammonium formate, 100 mM formic acid. If the samples were separated subsequently by paper chromatography, the following fractions were eluted [29]: fraction I (30 ml of H20); fraction II containing mosrtol monophosphates (20 ml of 200 mM ammonium formate, 100 mM formic acid); fraction III containing inositol bisphosphate (20 ml of 400 mM ammonium formate, 100 mM formic acid); fraction IV containing inosrtol trisphosphate (12 ml of 1 M ammonium formate, 100 mM formic acid). In order to remove the ammonium ions the eluates were passed through Dowex HCR-W2 columns (10 ml) followed by rinsing with 50 ml of H20. The eluates were then dried at 40°C by rotoevaporation ex vacua and further separated either by descending paper chromatography [20,21] or by HPLC. Separation by HPLC was carried out on a pBondapak NHz-column (3.9 mm x 30 cm) utilising a 20 min isocratic elution with 75 mM ammonium acetate/ acetic acid followed by a 120 min linear gradient to 2 M ammonium acetate/acetic acid, pH 4.0, at a flow rate of 1 ml/min. Fractions were collected, split into two halves and either measured for 3Hradioactivity by liquid scintillation counting or for

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phosphorus [30]. Inosttol phosphates in samples separated by paper chromatography were visuahsed by an ammomum molybdate spray [25]. The respective zones were cut out, eluted with 1 ml of 0.1 N NaOH and 1 ml of Hz0 and measured for 3H-radioactivity by liquid scintrllatron counting. 2.5. Separatron of mosltol phospholrplds Inositol phospholiplds were extracted at acidic pH [19] and separated on thm layer plates impregnated with 1% potassmm oxalate [31]. 3. RESULTS AND DISCUSSION The addition of thrombin to human platelets prelabeled with [3H]inositol induces the formation of four radioactive inositol phosphates: inositol 1-monophosphate, mosrtol 2-monophosphate, mositol 1,4-bisphosphate and inositol 1,4,5trisphosphate. These products were identified by cochromatography with authentic standards on HPLC or paper chromatography (fig.l,2). Both HPLC and paper chromatography separated isomers of inositol mono-, brs- and trisphosphate. Since the radioactive inositol phosphates formed in stimulated platelets cochromatographed with authentic standards in both chromatographic systems, it 1s evident that the formed inosrtol phosphates have the structures indicated above. One has to be aware, however, that the identification of any structure which IS based solely on cochromatography of radioactivity with authentic compounds is not absolute [32]. We observed that for the measurement of the various mosrtol phosphates one could not rely on the Dowex anion exchange column chromatography, since a small percentage of the bulk of [3H]inositol is eluted m the inositol monophosphate fraction (see also fig.la), and more importantly, between 10 and 25% of the inositol bisphosphate is eluted in the inositol trisphosphate fraction (unpublished). Since the amount of the radioactive mositol bisphosphate formed is high if compared to the amount of inositol trisphosphate, the use of the Dowex column chromatography would lead to false high levels for inositol trisphosphate. We therefore used the Dowex column chromatography only as first purification step of inositol phosphates before their final separation by HPLC or paper chromatography. Recovery of

a

LOOT

i

0. DS

”

RETENTION

TIME,

lx)

mm

Fig 1. HPLC-separation of mositol phosphates formed m human platelets after exposure to thrombin. Human platelets were prelabeled wtth [3H]mositol and etther sahne (a) or thrombm (2 U/ml) was added for 10 s (b) or 5 mm (c). The aqueous soluble [3H]mositol phosphates (M) were extracted, a mrxture of unlabeled mosrtol phosphate standards (A---A) was added, and the samples were purrfred on Dowex column chromatography before separatron on HPLC. For further detads see section 2.

unlabeled inosrtol phosphate standards after Dowex column chromatography and HPLC was 76 + 15010for inositol-1,4,5_trisphosphate, 66 f 15% for inositol 1,6bisphosphate, 69 f 9% for inositol 1-monophosphate and 72 + 8% for inositol 2-monophosphate (mean -t SD, n = 8). The formation of inositol bisphosphate and inositol trisphosphate can be detected 5 s after addition of thrombm (fig.3). Inositol trisphosphate reaches rts peak level at 10 s, whereas inositol brsphosphate accumulates further. In contrast, increased formation of both inositol l-monophosphate and inosltol 2-monophosphate is not detectable until 10 s after addition of thrombin. After 109

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Ftg 2. Formatton of mosnol i,4,5-trisphosphate m human platelets after exposure to thrombm Human platefets were prelabeled wnh [3~]lnosItol, and enher sahne (A---A) or thrombm (2 U/ml; o-----e) was added for 10 s, A mtxture of mosrtol phosphates produced by alkaline hydrolysis of phospho~nos~tldes [ZO]was added, pools of 5 samples were extracted and subjected to subsequent Dowex column chromatography [29] and descendmg paper chromatography [21]. The ftgure shows a radroactrve profde on descending paper chromatography of the Dowex-fractron contarmng mos1tol trisphosphate (lower panel) and the cochromatography wtth unlabeled mosttoi tr~sphosphat~ standard substances (upper panel)

Fig 3. Trme-course of mosttot phosphate formatron m human platelets after add&ton of thrombm Human platelets were prelabeled wrth r~~~lnos~tol Incorporation of ‘H mto phosphoinositldes of 0.9 ml platelet suspenston was 92691 cpm for PtdIns, 18835 cpm for PtdKns4P and 4490 cpm for PtdIns4,5Pz. Platelet suspensions were strmulated wrth thrombm (2 U/ml) for vartous times, a mrxture of unlabeled inosrtol phosphate standards was added and 3H-labeied mositoi phosphates were separated by subsequent Dowex column chromatography and HPLC as shown m fig I.

that time lag inosrtol I-monophosphate and mo&d 2-mono~hos~~ate show a co~tl~~o~s, large acc~m~~ati~n (fig.3, t&k I), The formation of inositoi Z-monophosphate m addition to mositof

I-monophosphate could reflect the tranaent formation of mositof t,2-cyclic-mo~op~osphate [27]. Similar trme courses of mos~tol phosphate formatlon upon specrfic cell activation have been

Thromb~n-~~du~~

Table 1 formatton of mosttol phosphates m human platelets

Additrons

None Thrombm, 10 s Thromb~n* 5 mm

3H-Radroactrvtty &pm) Ins Pi

Ins 1,4-Pz

Ins 1,4,5-P3

305 + 78 301 zt 63 6117 f 1953

24-+. 13 461 + 206 1653 t 299

1+ 2 5.5 4 27 44 + 22

Platelets prelabeied wrth 13Hfmosrtul were exposed to thrombm (2 U/ml). InosrtoI phosphates were separated by subsequent anion exchange cotumn chromatography and paper chromatography (see section 2) Values are mean + SD of 5 experiments

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reported for other cell types prelabeled with [3H]inositol ([33,34]; for ref. see [7]). The results indicate polyphosphoinositide hydrolysis, but also provide evidence for PtdIns hydrolysis m stimulated platelets. If the rate of conversron of labeled inositol phosphohpids to the corresponding inositol phosphates 5 min after addrtion of thrombm (2 U/ml) IS calculated, there IS a conversion rate of 1.5 + 0.6% for PtdIns4,5Pz, of 20 + 8% for PtdIns4P and of 10 + 3% for PtdIns (mean f SD from 3 experrments). This may indicate that there is only a small pool of radioactive PtdIns4JP2 - if compared to PtdIns4P and Ptdlns - which is degraded by phospholipase C. These results, however, do not exclude the pathway proposed recently by which Ptdlns is refueling PtdIns4P and PtdIns4,5Pz, which is then hydrolysed to inositol trisphosphate and degraded to mositol bisphosphate and monophosphate [7,18,29,33]. The rapid formatton of inositol 1,4-bisphosphate and mositol 1,4,5trisphosphate in stimulated platelets indicates that PtdIns4P and PtdIns4,5P2 are hydrolysed before PtdIns by phospholipase C. From the present results it seems that both PtdIns4P and PtdIns4,5P2 are degraded initially following platelet exposure to thrombm. The rapid, but restricted formation of the potent Ca’+-mobiliser inositol 1,4,5trisphosphate may indicate that the enzymatic hydrolyses of PtdIns4,5P2 IS tightly controlled. Another possible regulation step could be the actrvation of a specific phosphomonoesterase which degrades mositol 1,4,5_trisphosphate to inositol 1,6bisphosphate [35]. The study of the enzymatic regulation of the formation and inactivation of inositol 1,4,5-trisphosphate may give further Insights into the mechanism of platelet activation. ACKNOWLEDGEMENTS We are grateful to Ingeborg Drossel for the careful preparation of the manuscript and Professor Peter C. Weber for his kmd support of the study.

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1985

REFERENCES 111Lapetma, E.G. and Cuatrecasas, P. (1979) Btochum Biophys. Acta 573, 394-402. S. (1979) J. Chn. Invest. 63, 580-587. [31 Broekman, M.J., Ward, J.W. and Marcus, A J. (1980) J. Chn. Invest. 66, 275-283. 141 Lloyd, J V , Nishizawa, E.E. and Mustard, J.F. (1973) Br J. Haematol. 25, 77-99. 151 Siess, W , Siegel, F.L. and Lapetma, E.G (1983) J. Biol. Chem. 258, 11236-l 1242. Kl Siess, W., Bohhg, B., Weber, P.C. and Lapetma, E.G. (1984) Blood, in press [71 Berrrdge, M.J (1984) Btochem. J. 220, 345-360. PI Streb, H., Irvine, R.F., Berridge, M J. and Schulz, I (1983) Nature 306, 67-69. 191 Nrshrzuka, Y. (1983) Trends Btochem. Ser. 8, 13-16. 1101 Agranoff, B.W., Murthy, P. and Seguin, E.B. (1983) J. Btol. Chem. 258, 2076-2078. K., Takat, Y , Sawamura, M., 1111 Kaibuchr, Hoshilima, M., Fulikura, T. and Nishtzuka, Y. (1983) J. Biol. Chem. 258, 6701-6704. WI Stess, W., Cuatrecasas, P. and Lapetma, E.G. (1983) J Brol. Chem 258, 4683-4686. u31 Siess, W., Weber, P.C. and Lapetina, E.G. (1984) J. Blol. Chem. 259, 8286-8292. 1141 Siess, W., Siegel, F.L. and Lapetma, E.G. (1984) Biochrm. Btophys Acta, m press. WI Lapetma, E.G and Stess, W. (1983) Life Ser. 33, 1011-1018. iI61 Mrchell, R.H. (1975) Brochrm. Brophys. Acta 415, 81-147. 1171 Abdel-Latrf, A.A., Akhtar, R.A. and Hawthorne, J.N. (1977) Brochem J. 162, 61-73. 1181 Mrchell, R.H., Krrk, C.J., Jones, L.M., Downes, C. and Creba, J.A. (1981) Philos. Trans. R. Sot. London, Ser. B 296, 123-137. 1191 Billah, M.M and Lapetma, E.G. (1982) J. B~ol. Chem. 257, 12705-12708. WI Grado, C. and Ballou, C.E. (1961) J. Biol. Chem. 236, 54-60. WI Deslobert, A. and Petek, F. (1956) Bull. Sot Chum Brol. 38, 871-883. P21 Tomhnson, R.V. and Ballou, C.E. (1961) J. Biol. Chem. 236, 1902-1911 [23] Irvme, R F., Brown, K D. and Berrtdge, M.J. (1984) Btochem. J. 221, 269-272. 1241 Brllah, M.M. and Lapetina, E.G. (1982) J Brol. Chem 257, 5196-5200. [25] Sress, W. and Lapetma, E.G. (1983) Biochrm Brophys. Acta 752, 329-338, 1261 Mrchell, R.H. and Allan, D. (1975) FEBS Lett 53, 302-304

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[27] Plzer, F L and Ballou, C.E. (1958) J. Am Chem. Sot 85, 915-921. [28] Bligh, E.G and Dyer, W.J. (1959) Can J Btochem. Physlol. 37, 911-917 [29] Berrtdge, M.J , Dawson, R M C., Downes, D.P., Heslop, J.P. and Irvine, R.F (1983) Btochem J 212, 473-482 [30] Bartlett, G.R. (1959) J. Btol. Chem. 234, 466-468.

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[31] Jolles, J., Schrama, L H and &pen, W H. (1981) Brochtm. Brophys. Acta 666, 90-98 [32] Irvme, R.F , Letcher, A.J , Lander, D.J and Downes, C.P (1984) Biochem J. 223, 237-243. [33] Berrrdge, M.J. (1983) Btochem J 212, 849-858 [34] Thomas, A P , Alexander, J and Wtlhamson, J R (1984) J. Btol. Chem. 259, 5574-5584 1351 Downes, C.P , Mussat, M.C. and Mtchell, R H. (1982) Btochem. J 203, 169-177